Genome sequencing and comprehensive rare-variant analysis of 465 families with neurodevelopmental disorders.

Despite significant progress in unraveling the genetic causes of neurodevelopmental disorders (NDDs), a substantial proportion of individuals with NDDs remain without a genetic diagnosis after microarray and/or exome sequencing. Here, we aimed to assess the power of short-read genome sequencing (GS), complemented with long-read GS, to identify causal variants in participants with NDD from the National Institute for Health and Care Research (NIHR) BioResource project. Short-read GS was conducted on 692 individuals (489 affected and 203 unaffected relatives) from 465 families. Additionally, long-read GS was performed on five affected individuals who had structural variants (SVs) in technically challenging regions, had complex SVs, or required distal variant phasing. Causal variants were identified in 36% of affected individuals (177/489), and a further 23% (112/489) had a variant of uncertain significance after multiple rounds of re-analysis. Among all reported variants, 88% (333/380) were coding nuclear SNVs or insertions and deletions (indels), and the remainder were SVs, non-coding variants, and mitochondrial variants. Furthermore, long-read GS facilitated the resolution of challenging SVs and invalidated variants of difficult interpretation from short-read GS. This study demonstrates the value of short-read GS, complemented with long-read GS, in investigating the genetic causes of NDDs. GS provides a comprehensive and unbiased method of identifying all types of variants throughout the nuclear and mitochondrial genomes in individuals with NDD.

Sanchis-Juan A ，Megy K ，Stephens J ，Armirola Ricaurte C ，Dewhurst E ，Low K ，French CE ，Grozeva D ，Stirrups K ，Erwood M ，McTague A ，Penkett CJ ，Shamardina O ，Tuna S ，Daugherty LC ，Gleadall N ，Duarte ST ，Hedrera-Fernández A ，Vogt J ，Ambegaonkar G ，Chitre M ，Josifova D ，Kurian MA ，Parker A ，Rankin J ，Reid E ，Wakeling E ，Wassmer E ，Woods CG ，NIHR BioResource ，Raymond FL ，Carss KJ ... -  《-》

Combining optical genome mapping and RNA-seq for structural variants detection and interpretation in unsolved neurodevelopmental disorders.

Xiao B ，Luo X ，Liu Y ，Ye H ，Liu H ，Fan Y ，Yu Y ... -  《Genome Medicine》

Integrating RNA-Seq into genome sequencing workflow enhances the analysis of structural variants causing neurodevelopmental disorders.

Molecular diagnosis of neurodevelopmental disorders (NDDs) is mainly based on exome sequencing (ES), with a diagnostic yield of 31% for isolated and 53% for syndromic NDD. As sequencing costs decrease, genome sequencing (GS) is gradually replacing ES for genome-wide molecular testing. As many variants detected by GS only are in deep intronic or non-coding regions, the interpretation of their impact may be difficult. Here, we showed that integrating RNA-Seq into the GS workflow can enhance the analysis of the molecular causes of NDD, especially structural variants (SVs), by providing valuable complementary information such as aberrant splicing, aberrant expression and monoallelic expression. We performed trio-GS on a cohort of 33 individuals with NDD for whom ES was inconclusive. RNA-Seq on skin fibroblasts was then performed in nine individuals for whom GS was inconclusive and optical genome mapping (OGM) was performed in two individuals with an SV of unknown significance. We identified pathogenic or likely pathogenic variants in 16 individuals (48%) and six variants of uncertain significance. RNA-Seq contributed to the interpretation in three individuals, and OGM helped to characterise two SVs. Our study confirmed that GS significantly improves the diagnostic performance of NDDs. However, most variants detectable by GS alone are structural or located in non-coding regions, which can pose challenges for interpretation. Integration of RNA-Seq data overcame this limitation by confirming the impact of variants at the transcriptional or regulatory level. This result paves the way for new routinely applicable diagnostic protocols.

Riquin K ，Isidor B ，Mercier S ，Nizon M ，Colin E ，Bonneau D ，Pasquier L ，Odent S ，Le Guillou Horn XM ，Le Guyader G ，Toutain A ，Meyer V ，Deleuze JF ，Pichon O ，Doco-Fenzy M ，Bézieau S ，Cogné B ... -  《-》

Optical genome mapping unveils hidden structural variants in neurodevelopmental disorders.

While short-read sequencing currently dominates genetic research and diagnostics, it frequently falls short of capturing certain structural variants (SVs), which are often implicated in the etiology of neurodevelopmental disorders (NDDs). Optical genome mapping (OGM) is an innovative technique capable of capturing SVs that are undetectable or challenging-to-detect via short-read methods. This study aimed to investigate NDDs using OGM, specifically focusing on cases that remained unsolved after standard exome sequencing. OGM was performed in 47 families using ultra-high molecular weight DNA. Single-molecule maps were assembled de novo, followed by SV and copy number variant calling. We identified 7 variants of interest, of which 5 (10.6%) were classified as likely pathogenic or pathogenic, located in BCL11A, OPHN1, PHF8, SON, and NFIA. We also identified an inversion disrupting NAALADL2, a gene which previously was found to harbor complex rearrangements in two NDD cases. Variants in known NDD genes or candidate variants of interest missed by exome sequencing mainly consisted of larger insertions (> 1kbp), inversions, and deletions/duplications of a low number of exons (1-4 exons). In conclusion, in addition to improving molecular diagnosis in NDDs, this technique may also reveal novel NDD genes which may harbor complex SVs often missed by standard sequencing techniques.

Schrauwen I ，Rajendran Y ，Acharya A ，Öhman S ，Arvio M ，Paetau R ，Siren A ，Avela K ，Granvik J ，Leal SM ，Määttä T ，Kokkonen H ，Järvelä I ... -  《Scientific Reports》

Uncovering recessive alleles in rare Mendelian disorders by genome sequencing of 174 individuals with monoallelic pathogenic variants.

Clinical exome sequencing (ES) has facilitated genetic diagnosis in individuals with a rare genetic disorder by analysis of all protein-coding sequences in a single experiment. However, in 40-60% of patients, a conclusive diagnosis remains elusive. In 2-5% of these individuals, ES does identify a disease-associated monoallelic variant in a recessive disorder. We hypothesized that short-read genome sequencing (GS) might uncover a pathogenic variant on the second allele, thereby increasing diagnostic yield. We performed GS for 174 individuals in whom ES identified a monoallelic pathogenic variant in a gene associated with recessive disease related to their phenotype. GS interpretation was limited to the (non-)coding parts of the gene in which this first pathogenic variant was identified, focusing on splice-disrupting variants. Firstly, we uncovered a second pathogenic variant affecting coding sequence in five individuals, including two SNV/indel variants, two copy number variants, and one insertion. Secondly, for 24 individuals, we identified a total of 31 rare non-coding intronic SNV/indel variants, all predicted to disrupt splicing. Using functional follow-up assays, we confirmed an effect on splicing for three of these variants (in ABCA4, POLR3A and COL4A4) in three individuals. In summary, we identified a (likely) pathogenic second variant in 4.6% (8/174), and a possible diagnosis for 12.1% (21/174) of our cohort. Hence, when performing GS as first-tier diagnostic test, including the interpretation of SVs and rare intronic variants in known recessive disease genes, the overall diagnostic yield of rare disease will increase. The added diagnostic value of GS for recessive disease In our cohort of 174 individuals (84 males and 90 females) with a monoallelic pathogenic variant in genes associated with a wide and diverse range of recessive diseases (pie chart), using genome sequencing (GS) and a systematic approach (methods), we identified eight new diagnoses (4.6%). We identified a second likely pathogenic variant in eight individuals (results); In two a second coding variant was found, in three others, a rare non-coding SNV anticipated to disrupt splicing was uncovered, and in three individuals a structural rearrangement was identified (two copy number variants (CNV), and one structural variant (SV)).

Schobers G ，Pennings M ，de Vries J ，Kwint M ，van Reeuwijk J ，Corominas Galbany J ，van Beek R ，Kamping E ，Timmermans R ，Kamsteeg EJ ，Haer-Wigman L ，Cremers FPM ，Roosing S ，Gilissen C ，Kremer H ，Brunner HG ，Yntema HG ，Vissers LELM ... -  《-》